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Cited by 197Plant biochemistry and biosynthesisMicrobial Metabolic Engineering and BioproductionFungal and yeast genetics research

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Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid

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Ro, Dae-Kyun, Ouellet, Mario, Paradise, Eric M, Burd, Helcio, Eng, Diana, Paddon, Chris J, Newman, Jack D, Keasling, Jay D·2008·BMC Biotechnology
DOI 10.1186/1472-6750-8-83PMC PMC2588579
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Abstract

Background: Due to the global occurrence of multi-drug-resistant malarial parasites ( Plasmodium falciparum ), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood ( Artemisia annua ). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua . In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. Results: Three plant genes coding for amorphadiene synthase, amorphadiene oxidase ( AMO or CYP71AV1 ), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 μg mL -1 in shake-flask cultures and 1 g L -1 in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance ( PDR ) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast. Conclusion: The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.

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Figure 1 Amorphadiene production in the engineered yeast strain. (A) Plasmid harboring ADS under control of the Gal1 promoter used to produce amorphadiene in EPY300. Total (B) and specific (C) amorphadiene production from EPY300 expressing ADS on either pRS425-leu2 or pRS425-leu2d cultured in either CSM or YPG medium. Circles and diamonds indicate the leu2d and leu2 selection markers, respectively. Unfilled symbols indicate selective, minimal medium (CSM), and filled symbols indicate non-selective, rich medium (YPG). (D) Plasmid stabilities of yeast harboring pRS425-leu2::ADS or pRS425-leu2d::ADS grown in either CSM or YPG medium at 24, 72, and 144 hr post-induction. Data are mean ± standard deviation from three replicate experiments.
Figure 1 Amorphadiene production in the engineered yeast strain. (A) Plasmid harboring ADS under control of the Gal1 promoter used to produce amorphadiene in EPY300. Total (B) and specific (C) amorphadiene production from EPY300 expressing ADS on either pRS425-leu2 or pRS425-leu2d cultured in either CSM or YPG medium. Circles and diamonds indicate the leu2d and leu2 selection markers, respectively. Unfilled symbols indicate selective, minimal medium (CSM), and filled symbols indicate non-selective, rich medium (YPG). (D) Plasmid stabilities of yeast harboring pRS425-leu2::ADS or pRS425-leu2d::ADS grown in either CSM or YPG medium at 24, 72, and 144 hr post-induction. Data are mean ± standard deviation from three replicate experiments.CC BY · PubMed Central
Figure 2 Artemisinic acid production by the engineered yeast strain. (A) Plasmid encoding ADS, AMO, and CPR under control of either the Gal1 or Gal10 promoter used to produce artemisinic acid by EPY300. Total (B) and specific (C) artemisinic acid production from EPY300 strain expressing ADS, AMO, and CPR from either pESC-leu2 or pESC-leu2d cultured in either CSM or YPG medium. T indicates triple genes (ADS, AMO, and CPR). Circles and diamonds indicate leu2d and leu2 selection markers. Unfilled symbols indicate selective, minimal medium (CSM), and filled symbols indicate non-selective, rich medium (YPG). (D) Plasmid stabilities of yeast harboring either pRS425-leu2::ADS/AMO/CPR or pRS425-leu2d::ADS/AMO/CPR grown in either CSM or YPG medium at 24, 72, and 120-hr post-induction.
Figure 2 Artemisinic acid production by the engineered yeast strain. (A) Plasmid encoding ADS, AMO, and CPR under control of either the Gal1 or Gal10 promoter used to produce artemisinic acid by EPY300. Total (B) and specific (C) artemisinic acid production from EPY300 strain expressing ADS, AMO, and CPR from either pESC-leu2 or pESC-leu2d cultured in either CSM or YPG medium. T indicates triple genes (ADS, AMO, and CPR). Circles and diamonds indicate leu2d and leu2 selection markers. Unfilled symbols indicate selective, minimal medium (CSM), and filled symbols indicate non-selective, rich medium (YPG). (D) Plasmid stabilities of yeast harboring either pRS425-leu2::ADS/AMO/CPR or pRS425-leu2d::ADS/AMO/CPR grown in either CSM or YPG medium at 24, 72, and 120-hr post-induction.CC BY · PubMed Central
Figure 3 Plasmid stabilities of transgenic yeast strains. EPY307 harbors pESC-Leu2d-T plasmid and EPY309 harbors pESC-Leu2d-TKO, which carries AMO inactivated by the C439G point mutation.
Figure 3 Plasmid stabilities of transgenic yeast strains. EPY307 harbors pESC-Leu2d-T plasmid and EPY309 harbors pESC-Leu2d-TKO, which carries AMO inactivated by the C439G point mutation.CC BY · PubMed Central
Figure 4 Reverse-transcriptase PCR analysis of selected genes involved in multiple pleiotropic drug resistance. (A) Semi-quantitative RT-PCR conducted with RNA extracted after 24 hr of induction from EPY307 (artemisinic acid producer) and EPY309 (amorphadiene producer). Number of PCR Cycles used: ACT1: 23; PDR5, PDR15, SNQ2: 24; YOR1:27; PDR1:28; PDR3, PDR12:30. (B) Quantitative real-time RT-PCR conducted with samples harvested at 24 and 96 h post-induction.
Figure 4 Reverse-transcriptase PCR analysis of selected genes involved in multiple pleiotropic drug resistance. (A) Semi-quantitative RT-PCR conducted with RNA extracted after 24 hr of induction from EPY307 (artemisinic acid producer) and EPY309 (amorphadiene producer). Number of PCR Cycles used: ACT1: 23; PDR5, PDR15, SNQ2: 24; YOR1:27; PDR1:28; PDR3, PDR12:30. (B) Quantitative real-time RT-PCR conducted with samples harvested at 24 and 96 h post-induction.CC BY · PubMed Central
Figure 5 Heat-map representation of some up-regulated yeast genes identified by microarray analysis. Cluster 6 of k-mean clustering (k = 11) result showing some of the most strongly up-regulated genes observed in this study. Green color indicates the relative induction level (log2 ratio scale). S. cerevisiae open reading frame (ORF) and gene names are indicated. Red, blue, and black dots indicate genes for ATP-binding-cassette (ABC) superfamily, major facilitator superfamily (MFS), and oxidative stresses, respectively.
Figure 5 Heat-map representation of some up-regulated yeast genes identified by microarray analysis. Cluster 6 of k-mean clustering (k = 11) result showing some of the most strongly up-regulated genes observed in this study. Green color indicates the relative induction level (log2 ratio scale). S. cerevisiae open reading frame (ORF) and gene names are indicated. Red, blue, and black dots indicate genes for ATP-binding-cassette (ABC) superfamily, major facilitator superfamily (MFS), and oxidative stresses, respectively.CC BY · PubMed Central

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Authors
Ro, D.-K., Ouellet, M., Paradise, E. M., Burd, H., Eng, D., Paddon, C. J., Newman, J. D., & Keasling, J. D.
Affiliations
  • University of Calgary (CA)
  • University of California, Berkeley (US)
  • Amyris (United States) (US)
  • Lawrence Berkeley National Laboratory (US)
Corresponding author
Keasling, Jay D
ORCID
Venue
BMC Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2008-11-04
Volume
8
Issue
1
Pages
83-83
ISSN
1472-6750 (ISSN-L)
DOI
10.1186/1472-6750-8-83
PMC
PMC2588579
OpenAlex
W2112762465
Cited by
197
References
50
Citations by year
2026: 6 · 2025: 4 · 2024: 8 · 2023: 8 · 2022: 10 · 2021: 12 · 2020: 11 · 2019: 10 · 2018: 7 · 2017: 12 · 2016: 15 · 2015: 6 · 2014: 18 · 2013: 19 · 2012: 18
FWCI
4.53
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • BMC Biotechnology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Funders
Bill and Melinda Gates Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
Keywords
pleiotropic drug resistance, ABC transporters, NADPH-cytochrome P450 reductase, plasmid stability, oxidative stress response, osmotic stress response, gene expression regulation
MeSH terms
Polycyclic Sesquiterpenes, Animals, Antimalarials, Fermentation, Genetic Engineering, Plasmids, Prodrugs, RNA, Fungal, Saccharomyces cerevisiae, Sesquiterpenes, Gene Expression Regulation, Fungal, Genes, Plant, Point Mutation, Oxidative Stress, Oligonucleotide Array Sequence Analysis, Gene Expression Profiling, Drug Resistance, Multiple, Fungal, Artemisia annua, Artemisinins

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